CN108118027B - Sheep oocyte in-vitro 'two-stage' maturation method, and pre-incubation liquid and kit thereof - Google Patents
Sheep oocyte in-vitro 'two-stage' maturation method, and pre-incubation liquid and kit thereof Download PDFInfo
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Abstract
The invention discloses an in-vitro two-stage maturation method of sheep oocytes, a pre-incubation solution and a kit thereof, belonging to the field of embryo engineering. The pre-incubation solution includes oocyte in vitro basal medium TCM199 added with C-type natriuretic peptide and polyvinylpyrrolidone (PVP). Therefore, the C-type natriuretic peptide pre-incubation and in-vitro maturation combined 'two-stage' culture system provided by the invention can effectively promote the nuclear and cytoplasmic synchronous maturation of the sheep oocyte and improve the in-vitro developmental capacity of the oocyte, can provide high-quality egg sources and embryos for the production of the sheep in-vitro embryo, and better meets the requirements of scientific research and production.
Description
Technical Field
The invention relates to the field of embryo engineering, in particular to an in-vitro two-stage maturation method of sheep oocytes, a pre-incubation solution and a kit thereof.
Background
The in vitro maturation quality of the sheep oocyte is a key factor influencing in vitro fertilization and in vitro embryo development and also a leading factor restricting embryo transfer, embryonic stem cells and transgenic animal sources, so that the research on improving the in vitro maturation system of the oocyte becomes a breakthrough for improving the quality of the oocyte. Oocyte maturation in vitro proceeds through a multi-stage, complex, discontinuous process involving nuclear maturation, cytoplasmic maturation and granulocytic interaction with the oocyte.
Researchers have tried to simulate the physiological internal environment of oocyte survival and development, improve the oocyte in vitro maturation system, provide high-quality egg sources for production and scientific research, and further improve the embryo in vitro production efficiency. In 2008, the poplar like studied the influence of adding FCS with different concentrations in the basic culture solution for in vitro maturation of the sheep ovarian oocytes on in vitro maturation culture and in vitro embryo development, and the result found that adding FCS in the basic culture solution can significantly improve the in vitro maturation rate of the oocytes, and the influence difference of adding FCS with different proportions (10%, 15% and 20%) on the maturation rate of the oocytes is not significant. Meanwhile, the influence of different in vitro culture times on the maturation of the sheep oocytes is compared, the maturation rates of 24 h and 26h in vitro culture of the sheep oocytes (73.3 percent and 77.5 percent respectively) are obviously higher than the maturation rates of oocytes (62.5 percent and 65.0 percent respectively) of 18 h and 20 h in vitro culture, and the optimal in vitro maturation culture time is determined to be 24-26 h. (Yangxije et al. influence of culture conditions on in vitro maturation and fertilization of sheep ovarian oocytes [ J ]. proceedings of Ministry of agriculture in Gansu, 2008). In 2010, Guoblonghua researches that 100 ng/ml EGF is added into a sheep oocyte maturation liquid and an embryo development liquid and comparison of a polar body discharge rate, an embryo development capability and a blastocyst cell number shows that the EGF has a positive effect on both in-vitro maturation and embryo development of the sheep oocyte. (the effect of EGF on in vitro maturation, fertilization and embryonic development of ovine oocytes of different diameter follicles [ J ]. Shihe university 2010).
Zhang Meijia et al, found that the physiological paracrine factor C-type natriuretic peptide (CNP) produced by granulosa cells of the ovarian follicle wall layer of mice passes through the G protein coupled receptor NPR2 (NPPC receptor 2, NPR2) acting on the cumulus granulosa cells around the oocytes, its Receptor NPR2 is mainly expressed on cumulus Granulosa cells, activates Guanylate Cyclase (GC), causes an increase in the amount of cGMP synthesis, and cGMP is transported into oocytes through gap junctions between cumulus cells and oocytes to inhibit phosphodiesterase 3A (PDE 3A) activity, and maintains high levels of cAMP in oocytes to inhibit the recovery of meiosis in oocytes (Meijia Zhang, You-Qiang Su, Koji sugira, guliang Xia, and John j. eppig. Granulosa Cell Ligand NPPC and Its Receptor NPR2 main intain medical aridity arist in Mouse oyytes. science 2010). In 2014, Hiradate et al, Japanese scholars, also found that CNP plays an important role in inhibiting the process of oocyte nucleus meiosis recovery during in vitro culture of cumulus oocyte complexes of pigs (Hiradate Y, Hoshino Y, Tanemura K, Sato E. C-type biological peptide inhibitors of cells in cells of cells in vitro culture. Zygon. 2014). However, the effect and use of CNP on sheep oocytes is still unknown.
Although conventional in vitro maturation of sheep oocytes can enable the oocytes to complete meiosis in an in vitro environment, the synchronous maturation of nucleoplasm is difficult to achieve, and the production efficiency of in vitro embryos is influenced. Therefore, the simultaneous maturation of nucleoplasm becomes an urgent problem to be solved. In order to better simulate the in vivo environment, the in vitro maturation culture system of the sheep oocyte needs to be further optimized, and the requirements of scientific research and production are better met. Meanwhile, in the conventional in vitro culture process of oocytes, oocytes of medium-sized follicles of 3-6 mm are mostly adopted for testing or application. And are well known in the art: the oocytes of small follicles are considered to be oocytes with low developmental competence and poor quality, and are rarely applied to experiments or production, but the oocytes of small follicles have a large number of small follicles in ovaries, so that how to improve the in vitro developmental competence of the oocytes in the small follicles has important significance for exploring the reproductive potential of female animals, and the oocytes of small follicles are also a problem existing in the field.
Disclosure of Invention
In order to solve the problems in the field, the invention is based on the principle that C-type natriuretic peptide inhibits the meiotic recovery of oocytes, and provides a method for establishing a sheep oocyte "two-stage" in vitro maturation system (namely, the sheep oocytes are pre-incubated by using the pre-incubation liquid and then in vitro maturation culture is carried out by adding a maturation promoting mixture) by prolonging the maturation time of cytoplasm.
The technical scheme of the invention is as follows:
the invention provides a preincubation solution for in vitro maturation of sheep oocytes, which is characterized by comprising an oocyte in vitro basal medium TCM199 added with C-type natriuretic peptide and polyvinylpyrrolidone (PVP).
Further, the final concentration of the C-type natriuretic peptide in the oocyte in vitro basal medium TCM199 is 150 nM to 200 nM; and/or the presence of a gas in the gas,
the final concentration of the polyvinylpyrrolidone (PVP) in the oocyte in vitro basal medium TCM199 is 0.5 mg/ml-1.0 mg/ml.
In particular, the final concentration of the C-type natriuretic peptide in the oocyte in vitro basal medium TCM199 is selected from one of the following concentrations: 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM;
and/or the presence of a gas in the gas,
the final concentration of the polyvinylpyrrolidone (PVP) in the oocyte in vitro basal medium TCM199 is selected from one of the following concentrations: 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.
The invention provides a kit for in vitro maturation of sheep oocytes in the 2 nd aspect, which comprises the pre-incubation liquid.
Further, the kit also comprises conventional reagents for in vitro maturation of the sheep oocyte; for example, the conventional agent is a maturation-promoting agent; the maturation-promoting mixture comprises 20% FBS, 0.2 IU/ml FSH, and 0.1 IU/ml LH by volume.
In particular, the conventional reagent can also be a conventional in vitro maturation liquid; the conventional in vitro maturation solution comprises: TCM199, FBS 10% by volume, 0.05IU/ml FSH, 0.05IU/ml LH, 1ug/ml E2100U/mL penicillin, and 100U/mL streptomycin.
In a third aspect, the invention provides a method for in vitro 'two stage' maturation of ovine oocytes, comprising: pre-incubating the cells to be treated by adopting the pre-incubation liquid; and/or performing in-vitro maturation treatment on the cells to be treated by adopting the kit.
In specific embodiments, the pre-incubation comprises: placing the cells to be treated in the pre-incubation solution for culturing for 4 h; the aim of the pre-incubation is to improve the quality of cytoplasmic maturation of the sheep oocytes;
preferably, the pre-incubation further comprises: washing the cells to be treated 3 times with the pre-incubation solution before the culturing is carried out for 4 h; the effect of this step is to better adapt the cultured cells to the pre-incubation environment.
In a further embodiment, the pre-incubation further comprises: the pre-incubation was pre-incubated with CO before the incubation was carried out for 4h2Balancing in an incubator for more than 2 hours; the effect of this step is to bring the gas atmosphere of the pre-incubation close to the gas atmosphere of the incubator.
Further preferably, the culture conditions for 4h of culture are 5% CO2The temperature of the air is 38.6 ℃, and the saturated humidity is high.
Still further, the in vitro 'two-stage' maturation method of the sheep oocyte further comprises the following steps: after the pre-incubation, directly adding a maturation-promoting mixture for in vitro maturation culture; the effect of the maturation promoting mixture is to restore the meiosis process of the oocyte to achieve the final synchronous maturation of the nucleoplasm;
preferably, the in vitro maturation culture time is 26h, and the culture conditions are as follows: the culture environment was 38.5 ℃, 5% CO 2: 95% air, saturated humidity;
more preferably, the maturation-promoting agent is pre-CO-preceded before addition2Balancing in an incubator for more than 2 hours; the function of this step is to bring the gaseous environment that contributes to the maturation agent close to that of the incubator;
further preferably, the cell to be treated refers to a cumulus oocyte complex with uniform cytoplasm and relatively intact cumulus cell.
The invention provides a C-type natriuretic peptide pre-incubation liquid for inhibiting meiosis of a sheep oocyte nucleus. The final concentration of 150-200nM CNP and 0.5-1.0 mg/ml PVP were added to the oocyte in vitro basal medium TCM 199.
The invention also provides a novel method for the in vitro 'two-stage' maturation of the sheep oocyte. The C-type natriuretic peptide pre-incubation liquid is used for pre-incubating the sheep oocyte for 4 hours, and then a maturation promoting mixture is added for in-vitro maturation culture for additional 26 hours. The method can prolong the time of cytoplasm maturation, promote the oocyte to accumulate abundant maternal mRNA and protein, and achieve the synchronous maturation of nucleoplasm as far as possible.
In some embodiments of the invention, the pre-incubation step is specificThe cumulus oocyte compound which comprises uniform cytoplasm and relatively complete cumulus cells is washed 3 times by the C-type natriuretic peptide preincubation solution and then put into the pre-CO2Balancing the C-type natriuretic peptide pre-incubation liquid in an incubator for more than 2h, and culturing for 4 h; the culture conditions were: containing 5% CO2The temperature of the air is 38.6 ℃, and the saturated humidity is high. The in vitro maturation culture is to pre-treat with CO2Directly adding a maturation promoting mixture which is balanced in the incubator for more than 2 hours into the pre-incubation liquid for maturation culture for additional 26 hours; the culture environment was 38.5 ℃, 5% CO 2: 95% air, saturated humidity; the maturation promoting mixture comprises the following components: 20% (v: v) FBS +0.2 IU/ml FSH + 0.1 IU/ml LH.
In the invention, the C-type natriuretic peptide pre-incubation liquid can inhibit early recovery of meiosis, relatively prolong the maturation time of cytoplasm, promote the synthesis and accumulation of maternal mRNA and protein of oocyte, and promote the synchronous maturation of nucleoplasm to a certain extent, thereby improving the in vitro maturation quality of oocyte.
In vitro embryo production experimental data show that compared with the conventional in vitro maturation method, the embryo blastocyst rate and the blastocyst quality obtained by the in vitro 'two-stage' maturation system are obviously improved, the 'two-stage' in vitro maturation system can obviously improve the oocyte development capability of the sources of small follicles, and the utilization efficiency of the follicles on the sheep ovaries is improved.
In conclusion, the in vitro 'two-stage' maturation system constructed by the C-type natriuretic peptide and the PVP effectively improves the in vitro developmental capacity of the sheep oocyte. Compared with other chemical meiosis inhibitors such as Roscovitine, hypoxanthine, butyrolactone I, phosphodiesterase 3 and 6-DMAP, the C-type natriuretic peptide is non-toxic and harmless to oocytes and embryos, safe and effective, and is more beneficial to healthy development of the embryos. By adopting the CNP-pre-incubation two-stage in-vitro maturation system for pre-incubating the sheep COCs for 4h and then in-vitro maturation for 26h, the maturation quality of oocytes and the developmental capacity of later-stage embryos are both remarkably improved, and the blastocyst developmental rate cultured by adopting the two-stage in-vitro maturation pre-incubation liquid, the kit or the method is improved by at least about 10-20%. More importantly, the 'two-stage' maturation method, the pre-incubation solution or the kit has a very obvious effect of improving the in vitro developmental capacity in the aspect of improving the small follicles, and can ensure that the cleavage rate and the blastocyst rate of the in vitro mature embryos of the small follicles with the follicle diameter of less than 3.0 mm respectively reach 71.94 percent and 30.19 percent. Therefore, the double-stage in vitro maturation system can obviously improve the in vitro development capability of the sheep oocyte, is expected to be widely used in embryo in vitro production and scientific research, and has good application prospect in sheep embryo in vitro production.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments, but the scope of the present invention is not limited thereto. Unless otherwise specified, the procedures used in the following examples are conventional, and all reagents used are commercially available.
Main instrument equipment
CO2 incubator, constant temperature stage, stereomicroscope, super clean bench.
Primary reagent
FSH (Follicle stimulating hormone), LH (Luteinizing hormone), E2 (Estradiol), FBS (Fetal bovine serum), TCM199, PVP (polyvinylpyrrolidone), SOF broth (formulation see attached table 2), all purchased from Sigma.
The main biological material
Sheep oocytes were obtained from the ovaries of slaughterhouses.
Group 1 example, Pre-incubation of the invention
The group of embodiments provides a pre-incubation solution for in vitro maturation of sheep oocytes. All embodiments of this group have the following common features: the pre-incubation liquid comprises oocyte in vitro basal medium TCM199 added with C-type natriuretic peptide and polyvinylpyrrolidone (PVP).
In some specific embodiments, the final concentration of the C-type natriuretic peptide in the oocyte in vitro basal medium TCM199 is between 150 nM and 200 nM.
In other embodiments, the final concentration of polyvinylpyrrolidone (PVP) in the oocyte in vitro basal medium TCM199 is between 0.5 mg/ml and 1.0 mg/ml.
In a further embodiment, the final concentration of the C-type natriuretic peptide in the oocyte in vitro basal medium TCM199 is selected from one of the following concentrations: 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM; and/or the presence of a gas in the gas,
in other further embodiments, the final concentration of polyvinylpyrrolidone (PVP) in the oocyte in vitro basal medium TCM199 is selected from one of the following concentrations: 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.
In more specific examples, the specific formulation of the incubation liquid is shown in table 1 below:
the C-type natriuretic peptide refers to: c-type natriuretic peptide (CNP) or C-type natriuretic peptide, which have the meaning conventionally understood by those skilled in the art.
Polyvinylpyrrolidone (PVP) has a meaning commonly understood by those of ordinary skill in the art.
In a particular embodiment, the oocyte in vitro basal medium TCM199 is commercially available, for example, directly from Sigma or Gibco.
Group 2 examples, kits of the invention
The group of embodiments provides a kit for in vitro maturation of ovine oocytes. All the embodiments of this group have the following common features: the kit comprises a pre-incubation solution according to any of the embodiments of group 1.
In a further embodiment, the kit further comprises conventional reagents for in vitro maturation of ovine oocytes; for example, the conventional agent is a maturation-promoting agent; the maturation-promoting mixture comprises 20% FBS, 0.2 IU/ml FSH, and 0.1 IU/ml LH by volume.
In this particular set of embodiments, the conventional reagent may also be a conventional in vitro maturation liquid; the conventional in vitro maturation solution comprises: TCM199, FBS 10% by volume, 0.05IU/ml FSH, 0.05IU/ml LH, 1ug/ml E2100U/mL penicillin, and 100U/mL streptomycin.
EXAMPLE 3 group 3 maturation method of the invention
The present group of embodiments provides a method for in vitro "two stage" maturation of ovine oocytes. All embodiments of this group share the following common features: the maturation method comprises the following steps: pre-incubating cells to be treated with the pre-incubation solution of any of the embodiments of group 1; and/or, performing in vitro maturation on the cells to be treated by using the kit of any of the group 2 examples.
In a particular embodiment of this group, the pre-incubation comprises: placing the cells to be treated in the pre-incubation solution for culturing for 4 h; the aim of the pre-incubation is to improve the quality of cytoplasmic maturation of the sheep oocytes;
in a preferred embodiment of this group, the pre-incubation further comprises: washing the cells to be treated 3 times with the pre-incubation solution before the culturing is carried out for 4 h; the effect of this step is to better adapt the cultured cells to the pre-incubation environment.
In some more preferred embodiments of this group, the pre-incubation further comprises: the pre-incubation was pre-incubated with CO before the incubation was carried out for 4h2Balancing in an incubator for more than 2 hours; the effect of this step is to bring the gas atmosphere of the pre-incubation close to the gas atmosphere of the incubator.
In a further preferred embodiment, said culturing for 4h is carried out under conditions comprising 5% CO2The temperature of the air is 38.6 ℃, and the saturated humidity is high.
In a further embodiment, the maturation method further comprises: after the pre-incubation, directly adding a maturation-promoting mixture for in vitro maturation culture; the effect of the maturation promoting mixture is to restore the meiosis process of the oocyte to achieve the final synchronous maturation of the nucleoplasm;
preferably, the in vitro maturation culture time is 26h, and the culture conditions are as follows: the culture environment was 38.5 ℃, 5% CO 2: 95% air, saturated humidity;
more preferably, the maturation-promoting agent is pre-CO-preceded before addition2Balancing in an incubator for more than 2 hours; the function of this step is to bring the gaseous environment that contributes to the maturation agent close to that of the incubator;
further preferably, the cell to be treated refers to a cumulus oocyte complex with uniform cytoplasm and relatively intact cumulus cell.
Experimental example, concrete operation and effect verification of in vitro maturation of sheep oocyte
1. Maturation culture of sheep oocytes
The sheep oocyte is matured in vitro by the conventional method and the in vitro 'two-stage' method, and the method comprises the following steps:
1) conventional in vitro maturation:
step 1, collection of oocytes: using a sterile 10 mL syringe 12#A follicle with the diameter of 3-6 mm is extracted from the surface of an ovary by a needle, 1-2 mL of ovum collecting liquid is extracted In advance In a syringe before ovum collection, and Cumulus Oocyte Complexes (COCs) with uniform cytoplasm and relatively complete Cumulus cells are selected under a stereoscopic microscope and used for In Vitro Maturation (IVM).
Step 2, oocyte maturation culture: selecting a cumulus oocyte complex with uniform cytoplasm and relatively intact cumulus cells, and placing the cumulus oocyte complex into a conventional in vitro maturation solution (TCM 199 + 10% (v/v) FBS + 0.05IU/ml FSH + 0.05IU/ml LH + 1ug/ml E)2+ 100U/mL penicillin + 100U/mL streptomycin) for 24 h maturation in vitro at a culture density of 25/100 uL. The culture environment was 38.5 ℃, 5% CO 2: 95% air, saturated humidity.
2) "two stage" maturation: the oocyte preparation method is as follows:
step 1, collection of oocytes: using a sterile 10 mL syringe 12#A follicle with the diameter of 3-6 mm is extracted from the surface of an ovary by a needle, 1-2 mL of ovum collecting liquid is extracted In advance In a syringe before ovum collection, and Cumulus Oocyte Complexes (COCs) with uniform cytoplasm and relatively complete Cumulus cells are selected under a stereoscopic microscope and used for In Vitro Maturation (IVM).
Step 2, pre-incubating sheep oocytes with CNP: the selected COCs are washed 2-3 times by using the pre-incubation solution in any one of the group 1 embodiment and/or the pre-incubation solution in the kit in any one of the group 2 embodiment, and then transferred to the pre-incubation solution which is balanced for 2-4 h in a CO2 incubator for 4h, wherein the specific components of the pre-incubation solution are shown in the table 1 in the above embodiment.
The culture density is 25 pieces/50 uL culture environment is 38.5 ℃, 5% CO 2: 95% air, saturated humidity.
And 3, mature culture of the oocyte: 50uL of maturation-promoting mixture previously equilibrated in a CO2 incubator for 2-4 h was added directly to the CNP preincubation droplets for an additional 26h of in vitro maturation culture, the maturation-promoting mixture components being: 20% (v: v) FBS +0.2 IU/ml FSH + 0.1 IU/ml LH. The culture density is 25 pieces/100 uL, the culture environment is 38.5 ℃, and the culture environment is 5% CO 2: 95% air, saturated humidity.
2. In vitro fertilization of sheep oocytes
Thawing frozen sheep sperms at 39 ℃, gently adding thawed semen into the bottoms of 2 centrifuge tubes which are balanced for 2h and contain 1mL of upstream fluid (receiving semen), placing the tubes into an incubator for 45min at the upstream, sucking the upper semen and transferring the upper semen into a 1.5 mL centrifuge tube, centrifuging for 4 min at 1400 r/min, discarding the supernatant, adding 1mL of fertilization fluid and centrifuging again, discarding most of the supernatant, reserving about 100 mu L, gently blowing and uniformly mixing semen sediment, counting the sperms, and adding diluted sperm suspension into fertilization liquid drops to incubate with oocytes according to the sperm density requirement. The receptor fluid is: SOF + 2% oestrus sheep serum +6 IU/ml heparin sodium.
TABLE 2 composition of SOF culture broth
While incubating the sperm, the oocytes that were conventionally matured in vitro and provided for in vitro "two stage" maturation according to the present invention were washed in 0.1% hyaluronidase solution, respectively, to remove a portion of the cumulus cells. The oocytes were washed 3 times with the fertilization solution, and then 20 mature oocytes were placed in 50. mu.l of the fertilization drop.
3. In vitro culture of sheep embryos
After the sperms and the eggs are incubated for 20 h, the fertilized eggs are transferred into embryo culture solution, the culture solution is SOF + BSA, 50 fertilized eggs are transferred into a four-hole culture plate of 500 mul culture solution, and the culture condition is 5% CO2，5% O2，90%N2The temperature was 38.6 ℃. Embryos were examined 48 h and 144 h post fertilization for cleavage rate, blastocyst rate and extent of fragmentation of blastocyst nuclear DNA, respectively. The results are detailed in table 2.
4. Blastocyst quality detection
To assess blastocyst quality, the present invention compares the presence of fragmented blastocyst DNA obtained from oocytes from two maturation methods. The two-stage in vitro maturation, namely CNP preincubation for 4h and then in vitro maturation for 26h to obtain blastocysts, and conventional 24 h mature oocytes to obtain blastocysts are subjected to TUNEL and PI staining co-staining respectively. The results are detailed in Table 3.
TABLE 3 Effect of the Pre-incubation solutions of the present invention on the embryo development ability and blastocyst development quality of sheep oocytes after maturation and fertilization
Note: the data A and B in the same column in the table indicate that the difference is extremely significant (P<0.01, a and b represent significant differences (A)P<0.05）
As can be seen from Table 2, the blastocyst rate (58.91%) of the in vitro embryo of the "two-stage" in vitro maturation system obtained by pre-incubating COCs for 4h and then in vitro maturation for 26h with CNP is significantly higher than that of the in vitro embryo of the "two-stage" in vitro maturation system obtained by pre-incubating COCs for 4h with CNP（P<0.05) embryos obtained by conventional maturation for 24 h in vitro maturation (34.52%); the cleavage rate of the in vitro embryo of the "two-stage" in vitro maturation system (96.31%) was significantly higher than that of the conventional maturation group (82.62%,P<0.05). Furthermore, as can be seen from table 2, the "two-stage" in vitro maturation system in vitro blastula nuclear DNA fragmentation (6.19%) of CNP pre-incubation COCs for 4h followed by in vitro maturation for 26h according to the invention was very significantly lower (P) than<0.01) conventional maturation 24 h blastocysts (16.27%) obtained by in vitro maturation. The sheep oocyte cultured by the 'two-stage' in vitro maturation system is prompted to have stronger developmental capacity.
The embryo development ability and blastocyst development quality of the sheep oocyte obtained by adopting the pre-incubation liquid of any one group of the formula in the table 1 can obtain the cleavage rate, the blastocyst rate and the blastcyst nucleus DNA fragmentation rate similar to the data shown in the table 3, the difference between the upper value and the lower value of each group is not more than 0.1, and the details are not repeated for saving the text.
A "two stage" maturation method to improve the in vitro developmental competence of oocytes from small oocytes
In the present case, oocytes in small follicles with a follicle diameter of less than 3.0 mm are also cultured, and the preparation method of the oocytes is the same as the steps 1 to 3 in experimental example 2. The in vitro fertilization and in vitro culture method of the sheep oocyte is the same as 2-3 of the experimental example 2. The results are shown in Table 3.
TABLE 4 Effect of the "two-stage" maturation method of the present invention on the in vitro developmental Capacity of CoCs derived from Small oocytes
Note: in the table, different superscripts on the same column indicate significant differences (P < 0.05)
As can be seen from table 4, the embryo cleavage rate, blastocyst rate (71.94% and 30.19%, respectively) obtained by culturing small follicle (< 3.0 mm) derived oocytes in the "two-stage" in vitro maturation system of CNP pre-incubation COCs for 4h followed by in vitro maturation for 26h of the present invention were significantly higher (P < 0.05) than those obtained by conventional maturation of 24 h group of small follicle derived oocytes (59.62% and 19.68%, respectively). Has obvious promotion effect on the in vitro development of oocytes from small follicles.
The small oocysts obtained by treating the pre-incubation liquid of any one group of formulas in table 1 have cleavage rates and blastocyst rates similar to those shown in table 4 for each index related to embryo development ability, and the difference between the upper value and the lower value of each group is not more than 0.1, so that details are not repeated herein for saving text.
Claims (10)
1. An in vitro 'two-stage' maturation method of oocytes of ovine oocytes, comprising: pre-incubating and in-vitro maturation culturing are carried out on cells to be treated by adopting a kit for in-vitro maturation of sheep oocytes; the kit for in-vitro maturation of the sheep oocyte comprises a pre-incubation liquid and a conventional reagent for in-vitro maturation of the sheep oocyte; the pre-incubation liquid is an oocyte in vitro basal medium TCM199 added with C-type natriuretic peptide with the final concentration of 150 nM-200 nM and polyvinylpyrrolidone with the final concentration of 0.5 mg/ml-1.0 mg/ml;
the pre-incubation comprises: placing the cells to be treated in the pre-incubation solution for culturing for 4 h; the in vitro maturation culture time is 26h, and the culture conditions are as follows: the culture environment was 38.5 ℃, 5% CO 2: 95% air, saturated humidity;
the ovine small follicle oocyte is an oocyte in a small follicle with a follicle diameter of <3.0 mm.
2. The in vitro "two-stage" maturation method of oocytes of ovine oocytes according to claim 1, wherein the final concentration of the C-type natriuretic peptide in the in vitro basal medium TCM199 of oocytes is selected from one of the following concentrations: 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM;
the final concentration of the polyvinylpyrrolidone in the oocyte in vitro basal medium TCM199 is selected from one of the following concentrations: 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml.
3. The in vitro "two-stage" maturation method of oocytes of ovine oocytes according to claim 1, wherein the conventional agent is a maturation-promoting agent; the maturation-promoting mixture comprises 20% FBS, 0.2 IU/ml FSH, and 0.1 IU/ml LH by volume.
4. The in vitro 'two-stage' maturation method of oocytes of ovine oocytes according to claim 1, wherein the conventional reagent may also be a conventional in vitro maturation solution; the conventional in vitro maturation solution comprises: TCM199, FBS 10% by volume, FSH 0.05IU/ml, LH 0.05IU/ml, E1 ug/ml2100U/mL penicillin, and 100U/mL streptomycin.
5. The in vitro "two stage" maturation method of oocytes of ovine oocytes according to claim 1, wherein said pre-incubation further comprises: the cells to be treated were washed 3 times with the pre-incubation before the incubation was performed for 4 h.
6. The in vitro "two stage" maturation method of oocytes of ovine oocytes according to claim 5, said pre-incubation further comprising: the pre-incubation was pre-incubated with CO before the incubation was carried out for 4h2And balancing in the incubator for more than 2 hours.
7. The in vitro 'two-stage' maturation method of oocytes of ovine oocytes according to claim 5, wherein the culturing is carried out for 4h under conditions of 5% CO2The temperature of the air is 38.6 ℃, and the saturated humidity is high.
8. The method for the in vitro "two-stage" maturation of oocytes of ovine oocytes according to any one of claims 1 to 3 and 5 to 7, further comprising: after the pre-incubation, in vitro maturation culture is performed by directly adding a maturation-promoting agent.
9. According to the claimsThe in vitro 'two-stage' maturation method of oocytes of ovine oocytes of claim 8, wherein the maturation-promoting mixture is pre-treated with CO before addition2And balancing in the incubator for more than 2 hours.
10. The in vitro "two-stage" maturation method of oocytes of ovine oocytes according to claim 1, wherein the cells to be treated refer to the cumulus oocyte complex with uniform cytoplasm and relatively intact cumulus cells.
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